We have previously demonstrated that topical application of adenosine A2A receptor agonists promotes more rapid wound healing both in diabetic and normal animals. Adenosine A2A receptor occupancy influences the behavior of most cell types involved in wound healing including macrophages, fibroblasts and endothelial cells. Our ongoing studies demonstrate: 1.) A2A receptor occupancy promotes fibroblast collagen production; 2.) topical application of an A2A receptor agonist promotes angiogenesis in vitro and in vivo and the molecular mechanisms that contribute to enhanced angiogenesis include stimulation of macrophage and endothelial cell production of vascular endothelial cell growth factor (VEGF) and diminished endothelial production of the anti-angiogenic factor thrombospondin I; 3.) tissue (tPA) but not urokinase plasminogen activator expression is required for A2A receptor-mediated promotion of wound healing; 4.) cytokines (TNF, IL-1 and interferon-gamma) and the receptor for advanced glycation endproducts (RAGE) modulate adenosine A2A receptor expression and function. To better understand how adenosine A2A receptor occupancy promotes wound healing we propose the following two aims: Specific Aim I. Effects of Adenosine A2A Receptor Occupancy on microvascular endothelial cells, fibroblasts and macrophages To further dissect the mechanisms by which A2A receptors promote wound healing and the intracellular signal transduction pathways involved adenosine A2A agonist effects on secretion of VEGF, thrombospondin I, tPA and annexin II (which binds tPA to cell surface) will be studied in macrophages and endothelial cells, collagen and protease production in fibroblasts. Signal transduction (src kinase, protein kinase A, cAMP, MAPKinases, Akt kinase, A2A receptor-mediated activation of brain derived neurotrophic factor (BDNF) receptor pathways) for these functions will be probed by use of specific inhibitors and assays for signal protein concentration, function and phosphorylation. Specific Aim II: Receptor crosstalk in the promotion or inhibition of wound healing The effect of adenosine A, receptor, cytokine (TNF, 1L-1, Interferon-gamma) receptor and RAGE ligation on adenosine A2A receptor mRNA, protein and function as well as adenosine A2A receptor desensitization mechanisms (GRK2 recruitment to the plasma membrane) will be tested both in vitro and in vivo (RAGE and A1 KO mice). These studies should provide new insights into the mechanism by which adenosine A2A receptor agonists, a novel therapy undergoing clinical trials for wound healing of diabetic foot ulcers, promote wound healing.